This study was approved by the Chang Gung Medical Foundation Institutional Review Board (100-4358A3). The present study group consisted of 339 male patients diagnosed with primary OSCC who were admitted to Chang Gung Memorial Hospital, Lin-Kou, Taiwan, between 1999 and 2011. All cases were histologically confirmed and scored according to the recommendations for the reporting of specimens containing oral cavity, oropharynx and hypopharynx neoplasms by the Associations of Directors of Anatomical and Surgical Pathology (ADASP).[20] All patients signed informed consent for participation and were interviewed in a uniform manner before surgery by a well-trained interviewer. The questionnaire used in the interview sought detailed information on general demographics, as well as current and past cigarette smoking history, alcohol drinking and AQ chewing habits. Tumor tissue was collected, frozen immediately after excision in liquid nitrogen and stored at -80°C until DNA extraction. High-molecular weight DNA was purified as previously described [21]. All tumor specimens and tissue sections were retrieved from an archive.

FADD gene copy number was analyzed using pre-designed TaqMan copy number assay (Hs01625513_cn) (Applied Biosystems, Foster City, CA, USA) by qPCR on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in our laboratory. The qPCR analysis was performed according to the MIQE guidelines [22]. The hydrolysis probe to determine the copy number of the FADD gene is located within exon 2. The reference probe targets a copy-number neutral region of the RNase P gene, serving as an internal standard. The quantitative duplex PCR assay was carried out in a 96-well optical plate with a total volume of 10 μl per well. The reactions included 2 μl of gDNA (~10 ng), 0.5 μl of FADD TaqMan Copy Number Assay solution (20×), 0.5 μl of RNase P TaqMan Copy Number Reference Assay solution (20×), and 5 μl of TaqMan Genotyping Master Mix (2×) with the final volume adjusted with sterile water. All reactions were performed in triplicate. Thermal cycling conditions included initial denaturation at 95°C for 10 mins, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. The number of copies of the FADD gene was determined by relative quantitation (RQ) using the comparative Cq (ΔΔCq) method, which requires a healthy control sample (diploid) as a calibrator in all amplifications. The RNase P gene was co-amplified with the FADD gene and served as an internal standard. FADD gene copy number status was defined by a comparative Cq (ΔΔCq) > 0.59 indicating amplification [23].

Paraffin-embedded tumor sections (1.5μm) were deparaffinized in xylene and absolute alcohol and retrieved with heat in 10 mM citrate buffer (pH 6.0). Immunostaining was performed using the UltraVision Quanto Detection System HRP (Thermo Scientific, Cheshire, UK) and a Lab Vision Autostainer 360 (Thermo Scientific, Cheshire, UK). In brief, slides were treated with hydrogen peroxide block reagent (Thermo Scientific) for 10 mins and rinsed with phosphate-buffered saline (PBS). After blocking non-specific binding with Ultra V Block reagent (UltraVision Quanto Detection System HRP kit; Thermo Scientific, Cheshire, UK), tissue sections were incubated with rabbit polyclonal anti-FADD antibody (1:500) (H-181; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature. The tissue sections were then washed with PBS and incubated with a secondary antibody from the UltraVision Quanto Detection System HRP at room temperature, and subsequently visualized by reaction with diaminobenzidine (DAB) used as the chromogen substrate (DAB Quanto kit; Thermo Scientific, Cheshire, UK). Finally, the slides were counterstained with hematoxylin and coverslipped with Permount and examined for the extent and intensity of cytoplasm in tumor cells and for background staining by the pathologist (WYC) in a blinded manner. In the present study, the normal epithelium present in most samples showed cytoplasmic staining of the suprabasal layer. In carcinoma cells, FADD protein expression was found mainly in the cytoplasm and distributed homogeneously in most tumors. In tumors with strong cytoplasm staining, there was usually some accompanying nuclear staining. The scoring criteria are as reported in Gibcus et al.[17]. In brief, using the normal epithelium as a reference for normal expression levels, we scored all samples according to the intensity as 0, 1+, 2+ and 3+. We categorized the FADD staining as low FADD expression when the intensity was 0 and 1+ (Fig 1A and 1B), and high FADD expression when intensity was 2+ and 3+ (Fig 1C and 1D).

Statistical analysis was carried out using the Statistical Package for the Social Sciences (SPSS) version 13. The correlations between FADD gene copy number or protein expression status and clinical parameters were examined by the χ² test or Fisher’s exact test. Correlations between the FADD copy number and protein expression were tested with the χ² test. Survival curves were constructed by the Kaplan-Meier method, and the curves were compared using the log-rank test. The Cox regression model was applied to simultaneously adjust all potential prognostic variables, including age, primary tumor status, lymph node status and differentiation. The results were considered significant if P < 0.05.

Three hundred and thirty-nine patients with a diagnosis of OSCC were recruited into the study. The clinicopathological features of the patients are shown in Table 1. The most common primary sites were the bucca (44.8%, 152/339) and tongue (30.4%, 103/339). Overall, 85.6% (290/339) of the patients were cigarette smokers, 53.4% (181/339) were alcohol drinkers and 85.0% (288/339) chewed AQ. The primary treatment for these 339 patients was surgery; 232 (68.4%) patients underwent additional radiation therapy, and 89 (26.3%) underwent additional chemoradiotherapy. The median follow-up period was 60 months.

FADD copy number amplification was found in 69 (20.4%) OSCC patients (Table 2). The levels of FADD protein expression were categorized as low and high expression subgroups according to the intensity of cytoplasmic staining (Table 2). We determined that 146 (43.1%) tumors had low expression, and 193 (58.7%) had high expression. High FADD protein expression was accompanied by increased FADD gene copy number. Sixty (60/69, 87.0%) patients with FADD gene amplification showed high FADD protein expression (S1 Table).

As shown in Table 2, tumors with lymph node metastasis and lymph node extra-capsular spread (ECS) had a significantly higher frequency of FADD amplification (P ≤ 0.001). Similar results were observed for tumors with a high expression of FADD protein (P < 0.001). Furthermore, high FADD protein expression was associated with younger age at diagnosis (P = 0.034) and a higher grade of tumor differentiation (P = 0.011). We further analyzed the relationship between FADD and patient prognosis by univariate analysis (Fig 2). The results showed that FADD gene amplification was associated with poor overall survival (OS) (Fig 2B, P = 0.011) [hazard ratio (HR) = 1.527, 95% confidence interval (CI), 1.098–2.123] (Table 3). High FADD protein expression was associated with poor disease-free survival (DFS) (Fig 3A) (HR = 1.684, 95% CI, 1.209–2.345) and OS (Fig 3B) (HR = 1.387, 95% CI, 1.035–1.859). The patients with both FADD gene amplification and protein overexpression showed worse DFS (Fig 4A, P = 0.005) and OS (Fig 4B, P = 0.009). In addition to FADD, primary tumor status (HR = 1.676, 95% CI, 1.256–2.238) and lymph node metastasis (HR = 1.931, 95% CI, 1.310–2.846) were significantly associated with a poorer OS. Lymph node ECS was also significantly associated with a poorer DFS (HR = 3.124, 95% CI, 2.202–4.432). After adjusting for age at diagnosis, primary tumor status, lymph node status and tumor differentiation by multivariate Cox regression, patients with FADD gene amplification or high protein expression had the worst DFS (P = 0.028, HR = 1.483, 95% CI, 1.044–2.106) (Table 4). Of the FADD gene copy neutral cases, we observed that 49.3% (133/270) of cases displayed a high expression level of FADD protein independently of gene copy number alteration (S1 Table). High FADD protein expression was also associated with young age at diagnosis (P = 0.029) and lymph node metastasis (P = 0.025) in this subgroup (S2 Table). High FADD protein expression also was associated with DFS in this subgroup (HR = 1.690, 95% CI, 1.173–2.435) (S3 Table). After adjusting for age at diagnosis, primary tumor status, lymph node status and tumor differentiation using multivariate analysis, patients with high FADD protein expression also showed a poorer DFS (HR = 1.575, 95% CI, 1.078–2.300) (S4 Table).